Objective To develop a RT-PCR method for determination of Mouse Hepatitis Virus in Laboratory mice and Mongolian gerbils. Methods Two pair of primers specific to the sequences of S gene were designed according to the published Mouse Hepatitis Virus.With which the RNA of MHV was extracted and reversely transcribed to cDNA as a template for PCR amplification. The developed RT-PCR method was optimized, carries on the specificity, sensitivity, stability, repeatability test. And used for determination of 65 Mongolian gerbils and 12 mice.Results The developed RT-PCR method is good in specificity, ensitivity, stability, and repeatability; and its minimum detection limit using the recombinant plasmid containing MHV gene as atemplate was 3.1pg/μl,and the lowest detection virus titer is 10-3.ml-1.The 65 Mongolian gerbils after RT - PCR detection were negative;The 12 mice after RT - PCR detection, there were three MHV positive, compared with the MHV in Genebank, The homologies in nucleotide sequence of 3 positive mice all is 97%. Conclusion The developed RT-PCR method can be used in detecting the Mammalian Mouse HepatitisVirus (MHV) in laboratory animal ,such as mice and Mongolian gerbils.